Fig. 4: Impaired destabilization of the phospho-mimetic ID2 S5D mutant delays mitotic exit. | Nature Communications

Fig. 4: Impaired destabilization of the phospho-mimetic ID2 S5D mutant delays mitotic exit.

From: Regulated interaction of ID2 with the anaphase-promoting complex links progression through mitosis with reactivation of cell-type-specific transcription

Fig. 4

a HeLa cells expressing ID2 WT or S5 phospho-mutants were exposed to double thymidine block and analyzed by flow cytometry. Bar graph shows quantification of cells in G1, S, and G2/M in one representative experiment. Experiments were performed three times with similar results. b HeLa cells treated as in a were immunostained with pHH3 and analyzed by flow cytometry. Bar graph shows quantification of pHH3-positive cells. c Loss of regulation of ID2-S5D during S-G2/M transition. Western blot of HeLa cells expressing FLAG-ID2 WT or S5 phospho-mutants exposed to double thymidine block. d Exit from mitosis is delayed by expression of ID2-S5D mutant. Western blot of HeLa cells expressing FLAG-ID2 WT or ID2-S5D treated with RO3306. e Left panel, schematics of the cell synchronization protocol for the analysis of ubiquitylation of ID2 WT and phospho-mutants at the G2/M and M/G1 transition. Middle panel, western blot of HeLa cells co-expressing FLAG-ID2 WT or phospho-mutants and HA-ubiquitin immunoprecipitated with FLAG antibody. Right panel, western blot of whole cellular lysates (WCL). Molecular weight markers are indicated in kDa. Experiments were repeated two times with similar results. f HeLa cells expressing ID2 wild type or S5 phospho-mutants were transiently transfected with a plasmid expressing the E-box-luciferase cassette. Cells were synchronized by double thymidine block and analyzed for luciferase expression. Bar graph indicates means ± SD of three replicates. p-values are from unpaired t-test, unequal variance. Experiment was repeated three times with similar results. g Loss of binding to chromatin of E47 and HEB in cells overexpressing ID2 S5D. Western blot of chromatin fractions of HeLa cells transfected with ID2 WT or ID2-S5D, synchronized by double thymidine block, and collected at serial times after release. Experiments were repeated two times with similar results. In d, APC3 and Cyclin B1 are from the same blots; pHH3, CDH1 are from independent gels; in e, HA and FLAG are from different gels; in g, E47 and HEB/pHH3 are from different gel. Loading controls are from the same gel as FLAG except in e where α-tubulin is from the same gel as pHH3.

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